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Ocular graft-versus-host disease (oGVHD) is a fast progressing, autoimmunological disease following hematopoietic stem cell transplantation, leading to severe inflammation of the eye and destruction of the lacrimal functional unit with consecutive sight-threatening consequences. The therapeutic “window of opportunity” is narrow, and current treatment options are limited and often insufficient. To achieve new insights into the pathogenesis and to develop new therapeutic approaches, clinically relevant models of oGVHD are desirable. In this study, the ocular phenotype was described in a murine, chemotherapy-based, minor-mismatch GVHD model mimicking early-onset chronic oGVHD, with corneal epitheliopathy, inflammation of the lacrimal glands, and blepharitis. Additionally, corneal lymphangiogenesis was observed as part of oGVHD pathogenesis for the first time, thus opening up the investigation of lymphangiogenesis as a potential therapeutic and diagnostic tool.

Purpose: To compare the efficacy, safety, and tolerability of waterfree cyclosporine formulation (CyclASol) at 2 concentrations (0.1% and 0.05% of cyclosporine [CsA]) to vehicle when applied twice daily for 16 weeks in patients with dry eye disease (DED). An open-label Restasis (Allergan, Irvine, CA) arm was included to allow a direct comparison with an approved therapy. Design: An exploratory phase II, multicenter, randomized, vehicle-controlled clinical trial, double-masked between CyclASol and vehicle with an open-label comparator. Participants: Two hundred and seven eligible patients with a history of dry eye disease were randomized 1:1:1:1 to 1 of 4 treatment arms (CyclASol 0.05%, n = 51; CyclASol 0.1%, n = 51; vehicle, n = 52, and Restasis, n = 53). Methods: After a 2-week run-in period with twice-daily dosing of Systane Balance (Alcon, Fort Worth, TX), patients were randomized to the respective treatment arm and dosed twice daily for 16 weeks. Main Outcome Measures: The study was set up to explore efficacy on a number of sign and symptom end points including total and subregion corneal fluorescein staining, conjunctival staining, visual analog scale (VAS) for dry eye symptoms VAS severity, and Ocular Surface Disease Index (OSDI) questionnaire. Results: CyclASol showed a consistent reduction in corneal and conjunctival staining compared with both vehicle and Restasis over the 16-week treatment period, with an early onset of effect (at day 14). A mixed-effects model–based approach demonstrated that the CyclASol drug effect was statistically significant over vehicle (total corneal staining P < 0.1, central corneal staining P < 0.001, conjunctival staining P < 0.01). This model-based analysis suggests a significant CyclASol effect for OSDI as symptom parameter (P < 0.01). The numbers of ocular adverse events were low in all treatment groups. Conclusions: CyclASol showed efficacy, safety, and tolerability at 2 concentrations in moderate-to-severe DED. In a direct head-to-head against open-label Restasis, CyclASol was found to have an earlier onset of action, as early as after 2 weeks of treatment, in relieving the signs of DED, as measured by corneal and conjunctival staining. The central region of the cornea, an important area for visual function in dry eye sufferers, was shown to have the most benefit from treatment. Excellent safety, tolerability, and comfort profile supports this new CsA formulation as having a positive benefit-to-risk ratio.

Meibomian glands (MG) are large sebaceous glands located below the tarsal conjunctiva and the abnormalities of these glands cause Meibomian gland dysfunction (MGD) which is responsible for evaporative dry eye disease (DED). Accurate MG segmentation is a key prerequisite for automated imaging based MGD related DED diagnosis. However, Automatic MG segmentation in infrared meibography is a challenging task due to image artifacts. A deep learning-based MG segmentation has been proposed which directly learns MG features from the training image dataset without any image pre-processing. The model is trained and evaluated using 728 anonymized clinical meibography images. Additionally, automatic MG morphometric parameters, gland number, length, width, and tortuosity assessment were proposed. The average precision, recall, and F1 score were achieved 83%, 81%, and 84% respectively on the testing dataset with AUC value of 0.96 based on ROC curve and dice coefficient of 84%. Single image segmentation and morphometric parameter evaluation took on average 1.33 s. To the best of our knowledge, this is the first time that a validated deep learning-based approach is applied in MG segmentation and evaluation for both upper and lower eyelids.

Purpose: Meibomian gland disease is generally accepted as the leading cause for evaporative dry eye disease (DED). In a previous study, perfluorohexyloctane, a semifluorinated alkane, has been demonstrated to significantly increase tear film breakup time and to reduce corneal fluorescein staining in patients with evaporative DED, thereby vastly reducing dry eye-related symptoms. This study was set up to evaluate perfluorohexyloctane in a larger population of patients with Meibomian gland dysfunction. Methods: Seventy-two patients with Meibomian gland disease and associated dry eye received 1 drop of perfluorohexyloctane 4 times daily during an observational, prospective, multicenter, 6–8-week study. Clinical assessment included best-corrected visual acuity, intraocular pressure, Schirmer test I, tear film breakup time, anterior and posterior blepharitis assessment, number of expressible Meibomian glands, meibum quality and quantity, ocular surface fluorescein staining, lid margin and symptom assessment, and Ocular Surface Disease Index (OSDI©). Results: From the 72 patients recruited, 61 completed the trial per protocol. Nine patients did not apply the medication as recommended and 2 patients were lost to follow-up. Tear film breakup time, corneal and conjunctival fluorescein staining, number of expressible Meibomian glands, and severity of anterior and posterior blepharitis significantly improved after 6–8 weeks of perfluorohexyloctane application. In addition, symptoms improved as demonstrated by a significant decrease of OSDI-values from 37 (±13) to 26 (±16). Conclusions: In concordance with previous findings, 6–8 weeks of topical application of perfluorohexyloctane significantly improves clinical signs of Meibomian gland disease and associated mild to moderate DED.

Lymphangiogenesis is essential for fluid homeostasis in vascularized tissues. In the normally avascular cornea, however, pathological lymphangiogenesis mediates diseases like corneal transplant rejection, dry eye disease, and allergy. So far, a physiological role for lymphangiogenesis in a primarily avascular site such as the cornea has not been described. Using a mouse model of perforating corneal injury that causes acute and severe fluid accumulation in the cornea, we show that lymphatics transiently and selectively invade the cornea and regulate the resolution of corneal edema. Pharmacological blockade of lymphangiogenesis via VEGFR-3 inhibition results in increased corneal thickness due to delayed drainage of corneal edema and a trend towards prolonged corneal opacification. Notably, lymphatics are also detectable in the cornea of a patient with acute edema due to spontaneous Descemet´s (basement) membrane rupture in keratoconus, mimicking this animal model and highlighting the clinical relevance of lymphangiogenesis in corneal fluid homeostasis. Together, our findings provide evidence that lymphangiogenesis plays an unexpectedly beneficial role in the regulation of corneal edema and transparency. This might open new treatment options in blinding diseases associated with corneal edema and transparency loss. Furthermore, we demonstrate for the first time that physiological lymphangiogenesis also occurs in primarily avascular sites.

Introduction: Anti-inflammatory, topical therapy of severe keratitis in dry eye disease (DED) and ocular graft-versus-host disease (oGvHD) includes steroids, cyclosporine (Cs), and others. In Germany, a commercial product containing 0.1% Cs in a cationic formulation is available since 2015. Objective: The aim of this study was to present real-life data using cationic 0.1% Cs in oGvHD patients. Methods: This was a retrospective study of 26 oGvHD and 41 DED patients with corneal staining of at least Oxford grade III. Parameters analyzed were Ocular Surface Disease Index, corneal staining, intraocular pressure, tear film break-up time, Schirmer, and visual acuity. In addition, it was evaluated how different Cs formulations were tolerated. Results: Corneal staining improved significantly in 1 eye in DED but not in oGvHD. In DED, cationic 0.1% Cs was not tolerated by 32% of the patients, in contrast to 0.05% Cs in castor oil not tolerated by 47% and liposomal 0.05% Cs by 63%. In oGvHD patients, cationic 0.1% Cs was not tolerated by 62%, 0.05% Cs in castor oil by 33%, and liposomal 0.05% Cs by 39% of the patients. Conclusions: This study demonstrates differences between the tolerance of different Cs formulations depending on the underlying cause of severe keratitis. Cationic 0.1% Cs is considerably less tolerated in oGvHD, and its use should be considered with care.

Conjunctiva-associated tissue (CALT) is assumed to play a crucial role in the immune system of the ocular surface. Its function in several ocular surface diseases (OSD) is still not fully understood. This study investigates the function of CALT in mouse models of dry-eye disease and ocular allergy. Since antigen-presentation is the central similarity in the pathologies, this study focuses on antigen-presentation in CALT Morphology and the expression of CALT, which was investigated in mice after induction of dry-eye, ocular allergy, topical antigen-stimulation, and after local depletion of phagocytic cells. Antigen uptake was investigated after the application of fluorescent ovalbumin (OVA). OSD influences the appearance and morphology of CALT in a disease-dependent manner. Ocular allergy leads to an increase and dry-eye disease to a decrease in number and size of CALT. The development of CALT is dependent on the presence of APCs. Professional APCs are present in CALT, and soluble antigen is transported into the follicle. CALT appearance is disease-specific and indicative of differing functions. Although the specific involvement of CALT in OSD needs further study, the existence of functional APCS and antigen-uptake supports the hypothesis that CALT is an immunological key player at the ocular surface.

Background Meibomian Gland Dysfunction (MGD) is a leading cause of evaporative Dry Eye Disease (DED). This makes non-invasive meibography an important procedure in the clinical evaluation of DED patients. Our purpose was to conduct a lead-off investigation focused on the practicality of performing meibography in a developing country, with limited access to complex ophthalmic imaging systems, using a custom meibographer, as a step to future comparative studies on meibomian glands and DED in Africa. Methods Meibomian glands(MG) in 76 upper eyelids (UL) and 49 lower eyelids (LL) in 1 eye each of 125 patients randomly selected from a patient population presenting with subjective DED symptoms at a clinic were photographed using a custom meibographer. Single frames were captured, and the MG area determined by intensity threshold segmentation and area calculation using Image J software. MG loss (MGL) was quantified by outlining its area and expressing it as a percentage of the total MG per Pult’s grading scheme. Dry eye measures included Tear Film Break Up - Time (TUBT), Schirmer’s test and Ocular Surface Staining (OSS). Symptoms were evaluated using the SPEED II questionnaire. Correlations between MGL and age, ocular signs and symptoms were analyzed by Pearson’s. Differences between comparable groups were analyzed by Mann - Whitney test; p

Purpose: Ocular graft-versus-host disease (oGvHD) following allogeneic hematopoietic stem cell transplantation develops as severe dry eye disease (DED) and is initially treated with lubricants, although no clinical trials are available using artificial tears in oGvHD. This trial was set up to test perfluorohexyloctane (NovaTears®) as nonpreserved layer-forming agent for the treatment of DED in oGvHD. Methods: 25 patients with severe DED due to oGvHD received 1 drop perfluorohexyloctane 4 times daily during a prospective, multicenter, observational 12-week study on top of established topical therapy. Clinical parameters included Schirmer test, tear film breakup time, corneal staining, meibum secretion and ocular surface disease index. Adverse events, visual acuity and intraocular pressure were key safety parameters. Results: From 25 patients recruited, 23 presented for the second visit. Perfluorohexyloctane treatment did not lead to any changes in clinical or safety parameters but led to fast relief in symptoms in 57% of the patients. One adverse reaction occurred. Conclusions: This study showed no change in clinical signs in severe DED due to oGvHD, which was not unexpected due to the underlying pathomechanisms. However, the study showed improvement of symptoms in individual patients allowing application of perfluorohexyloctane as an additional symptomatic therapy in oGvHD.

Purpose Corneal neovascularization, in particular lymphangiogenesis, is a limiting factor in corneal transplant survival. Novel treatment approaches focus on (selective) inhibition and regression of lymphatic vessels. Imaging clinically invisible corneal lymphatic vessels is a prerequisite for these strategies. Using a murine model, this study investigates whether corneal lymphatic vessels can be imaged using microscopic optical coherence tomography (mOCT). Methods Corneal neovascularization was induced by intrastromal placement of 11.0 nylon sutures in one eye of BALB/c mice. After 2 weeks, cross-sectional images and volumes of the corneas with a 0.5 mm lateral and axial field of view were acquired using a custom-built mOCT system enabling a resolution of 1 μm at a B-scan rate of 165/s. Three of the six animals received an additional intrastromal injection of India ink 24 hours before the measurement to stain the corneal lymphatic system in vivo. Immunohistochemistry using CD31 and LYVE-1 was used to validate the mOCT findings. Results Using mOCT, lymphatic vessels were visible as dark vessel-like structures with the lumen lacking a hyperreflective wall and mostly lacking cells. However, individual, slowly moving particles, which most likely are immune cells, occasionally could be observed inside the lumen. In lymphatic vessels of ink-stained corneas, hyperreflection and shadowing underneath was observed. Ink-filled lymphatic vessels were colocalized in consecutive corneal flat mounts of the same specimen. Conclusions Corneal lymphatic vessels can be imaged using mOCT. This novel approach opens new options for noninvasive clinical imaging of corneal lymphatic vessels for diagnostic and therapeutic indications.

Purpose: Evaporation of the tear film is heavily discussed as one core reason for dry eye disease (DED). Subsequently, new artificial tear products are developed that specifically target this pathomechanism. Perfluorohexyloctane (F6H8, NovaTears®) from the family of semifluorinated alkanes is a novel substance that has been approved as a medical device, as a nonblurring wetting agent for the ocular surface. Methods: Thirty patients with hyperevaporative dry eye received F6H8 during a prospective, multicenter, observational 6-week study. Patients were advised to apply 1 drop 4 times daily in both eyes. Parameters assessed included best corrected visual acuity, intraocular pressure, Schirmer I test, tear fluid, tear film breakup time (TFBUT), corneal staining, meibum secretion, and Ocular Surface Disease Index (OSDI©). Results: From the 30 patients recruited, 25 completed the trial per protocol. Four patients discontinued F6H8 and 1 patient did not present for follow-up. F6H8 treatment led to significant reduction of corneal staining and significant increase of Schirmer I and TFBUT. In addition, OSDI score dropped significantly from a mean of 55 (±23.0) to 34 (±22.4). Visual acuity and ocular pressure did not change. Conclusions: This prospective observational study shows significant beneficial effects in patients suffering from evaporative DED, using F6H8 in all the relevant parameters tested. The decrease of the OSDI by a mean of 21 points was particularly remarkable and clearly exceeds minimal, clinical important differences for mild or moderate and severe disease. Overall, F6H8 (NovaTears) seems to be safe and effective in treating mild to moderate hyperevaporative DED.

Importance Descemet membrane endothelial keratoplasty (DMEK) is a challenging procedure for the surgeon, particularly because of deficient visibility of the delicate tissue due to the natural en face view through the operating microscope. A cross-sectional view would greatly enhance intraoperative overview and enable the surgeon to better control the procedure. Objective To retrospectively analyze the use of intraoperative optical coherence tomography (iOCT) for improving the safety of DMEK. Design Intraoperative OCT during DMEK was performed in 26 eyes of 26 patients. We retrospectively analyzed imaging and video data. Setting Department of Ophthalmology, University of Cologne. Participants Seven men and 19 women aged 39 to 93 years with corneal endothelial dysfunction undergoing DMEK. Exposure Descemet membrane endothelial keratoplasty. Main Outcomes and Measures Visibility of surgical steps, overall duration of DMEK, overall time for complete intraoperative air filling of the anterior chamber, and correlation between donor age and Descemet rolling behavior. Results Intraoperative OCT enables visualization of all steps of the DMEK procedure. Overall mean (SD) duration of the DMEK procedure was 25.7 (6.9) minutes when using iOCT. Overall mean (SD) complete intraoperative anterior chamber air-filling time was 236 (108) seconds in contrast to 60 to 90 minutes for standard air-filling time. Descemet membrane rolling behavior showed significant inverse correlation between donor age (range, 39-93 years) and the extent of rolling ( R 2 = 0.5 [ P = .006]). Conclusions and Relevance Intraoperative OCT enhances the visibility of graft orientation and unfolding, thereby improving safety of the DMEK procedure. Overall, iOCT is a helpful device that may support surgeons in all steps of DMEK procedures.

Ab interno glaucoma surgery, such as trabecular aspiration or ab interno trabeculotomy, has been introduced as an alternative, non-filtering procedure to lower intraocular pressure in glaucoma patients. The purpose of the present study was to assess the feasibility of intraoperative spectral domain optical coherence tomography (iOCT) for improving ab interno glaucoma surgery.Intraoperative optical coherence tomography was performed to visualize the anterior chamber angle, the aspiration canula during trabecular aspiration, and the trabectome device during ab interno trabeculotomy. A commercially available 840-nm iOCT was used in a clinical setting, a 1310-nm spectral-domain OCT in an experimental setting.In the clinical setting using the 840-nm iOCT, visualization of the aspiration canula and the trabectome device was possible, however, with rather limited visibility of the chamber angle and the trabecular meshwork. In the experimental setting, the 1310-nm OCT enabled excellent visualization of the chamber angle and detailed evaluation of the instrument-trabecular meshwork relation.Intraoperative use of optical coherence tomography may be helpful for ab interno glaucoma surgery. Adjustments of the commercially available iOCT, including longer wavelengths and oblique scanning options, are necessary for the clinical use.

To analyze potential alterations in corneal nerve morphology and function in different stages of Fuchs’ endothelial corneal dystrophy (FECD). Thirty eyes with FECD underwent in vivo confocal microscopy using the Heidelberg Retina Tomograph II (HRT II; Heidelberg Engineering, Heidelberg, Germany) and the Rostock Cornea Module (RCM) to quantify the morphology of the central subbasal corneal nerve plexus (total nerve length, total nerve number, number of main nerve trunks, number of nerve branches) as well as esthesiometry (using the Cochet-Bonnet esthesiometer) of the central cornea to determine central corneal sensation as a measure of nerve function. Findings were correlated with an age-matched control group of 30 healthy individuals. Comparisons to biomicroscopical stage of FECD, visual acuity and central corneal thickness were performed using Spearman correlation. Depending on slit-lamp examination, all 30 eyes were classified into FECD stage 1–4 (stage 1: six eyes; stage 2: 15 eyes; stage 3: six eyes; stage 4: three eyes). Total nerve length (ρ = −0.8, p

Correct early graft attachment is believed to be crucial for final visual outcome after Descemet membrane endothelial keratoplasty (DMEK). Nonetheless, it is not yet known which imaging technique gives superior results for examining early postoperative graft adherence status. We compared imaging data taken with two different OCT devices to examine the development of graft adherence immediately after DMEK and to determine the superior device in terms of visualization of graft adherence. Ten consecutive patients (1 man/9 women) were examined three times postoperatively within the first 7 h after DMEK surgery using spectral domain OCT (SD-OCT) and time domain OCT (TD-OCT), as prospective case series and retrospective image data analyses. The parameters analyzed were localization and number, visibility and size of graft detachments. TD-OCT was able to detect a greater number of graft detachments after DMEK; however, SD-OCT provided better resolution of minor detachments. Graft detachments varied in position and degree at different time points immediately after surgery. All patients had some graft detachment within the first 7 h after DMEK surgery. TD-OCT enabled better overall analysis of graft detachments, even in the periphery, whereas SD-OCT allowed for the detection of even minor detachments, which suggests that a combination of the two techniques is optimal. Our results indicate that dynamic processes affecting the DMEK graft immediately after transplantation are responsible for changes in the attachment of donor tissue at an early postoperative stage. Modulation of early graft attachment may improve the final graft attachment.

Vascular endothelial growth factor (VEGF) has been shown to play an important role during endochondral bone formation in hypertrophic cartilage remodeling, ossification, and angiogenesis, but it is not expressed in normal adult articular cartilage. Thyroid cartilage undergoes only partial ossification beginning at the age of about 20. Because it never completely ossifies, we investigated a possible role of VEGF and its receptors (VEGFRs) as well as the angiogenetic inhibitor endostatin in this permanent cartilage. In analysis of cartilage samples from all specimens evaluated, VEGF121 and VEGF165 were identified as the only VEGF splice forms expressed. In addition to VEGF, VEGFR-2 (kinase domain region/fetal liver kinase 1), but not VEGFR-1 (fms-like tyrosine kinase 1), was detectable by RT-PCR in cartilage. However, VEGFR-2 expression was only detectable up to the age of 19 years. Deposition of VEGF and VEGFR was confirmed by immunohistochemistry. VEGF concentrations measured by ELISA in thyroid cartilage increased with age in males but decreased in females. Endostatin concentrations measured by ELISA in thyroid cartilage were three times lower than in articular cartilage and showed no change with age, either in females or males. VEGF was immunostained within the intra- and pericellular matrices of some but not all chondrocytes. Thus, apart from its production in hypertrophic chondrocytes of growth plates, VEGF is also produced in single chondrocytes of thyroid cartilage. The data allow us to speculate that thyroid cartilage persists in an embryological state until it has reached its final size. After reaching its final size at the end of the second decade, VEGFR-2 is downregulated and ossification starts in the posterior part of the thyroid cartilage, proceeding ventrally. Both proteins, VEGF121 and VEGF165, should contribute to this process. VEGF concentration is high and changes in an age-related and sex-specific manner. Therefore, we postulate that VEGF is at least one of the key factors that is important for the lifelong ossification in thyroid cartilage.

The occurrence of primary extranodal marginal-zone B-cell lymphoma [of the mucosa-associated lymphoid tissue (MALT) type] has only been described in the supraglottic region, implying that preexistent organized lymphoid tissue is present at that site only. To date, studies have not established clearly whether organized MALT shows a site-related distribution in the larynx. The supraglottic region of the false vocal folds and the subglottis from 87 unselected cadavers with no known history of nasal, oral, pharyngeal, laryngeal, tracheal, or esophageal disease were examined for the presence of organized MALT. Organized lymphoid tissue was found with the cytomorphological and immunophenotypic features of MALT in 100% of false vocal folds of children and in more than 90% of adolescents, decreasing to 7.1% in persons in their sixth decade, whereas MALT was completely absent in the subglottis in all age groups. The results explain why primary extranodal marginal-zone B-cell lymphoma has only been described in the supraglottic region but is absent in the subglottis. Moreover, the results suggest a region-specific immunological response of the different laryngeal areas as reflected in clinical observations and animal studies. However, the impact on presence or absence of laryngeal MALT awaits clarification.

Recent studies have identified diminishment of corneal nerves as another hallmark of Fuchs endothelial corneal dystrophy. This study aimed to analyze changes in corneal nerves after Descemet membrane endothelial keratoplasty (DMEK).Twenty-five patients were assessed for nerve alterations preoperatively and 1 week, 4 months, and 20 months after DMEK surgery. Morphology of the central subbasal nerve plexus was quantified by in vivo confocal microscopy.The total nerve length (481.2 ± 81.9 vs. 1536.0 ± 123.8 μm per frame, P0.0001), total nerve number (2.2 ± 0.3 vs. 7.2 ± 0.5 per frame, P0.0001), number of main nerve trunks (1.8 ± 0.2 vs. 3.5 ± 0.3 per frame, P0.0001), and number of nerve branches (0.5 ± 0.2 vs. 3.7 ± 0.4 per frame, P0.0001) were significantly decreased 1 week after DMEK compared with preoperative measurements. Ten months postoperatively, corneal nerves recovered to preoperative values. Central corneal sensation significantly reduced postoperatively (5.1 ± 1.0 vs. 6.0 ± 0.0, P = 0.001), but recovered during follow-up (10 months: 6.0 ± 0.0).DMEK diminishes the density and the function of subbasal corneal nerves early after transplantation. However, a complete recovery of corneal nerve density and function up to preoperative values occurs within 4 to 10 months.

Meibomian gland loss in ocular GvHD was described as a mechanism contributing to dry eye and severe damage to the ocular surface. Infrared images of upper eyelid meibomian glands from 86 ocular GvHD patients, from 10 patients after allogeneic stem cell transplantation (aSCT) without ocular GvHD, from 32 patients prior to aSCT and from 30 healthy controls were analyzed retrospectively and evaluated using two grading schemes. The upper meibomian gland area (uMGA) was calculated and set in relation to the total tarsal area of the lid. Results demonstrate that meibomian gland loss is significantly increased in patients with ocular GvHD as well as in patients prior to aSCT in comparison with controls (P between 0.05 and

Background Peripheral corneal graft detachment after Descemet9s membrane endothelial keratoplasty (DMEK) is a frequently occurring postoperative complication. The natural course of these persistent peripheral detachments over time is not known. Methods 166 patients were surveyed by slit-lamp-adapted optical coherence tomography (SL-OCT) directly after surgery, during first postoperative week, 4 weeks, 3, 6 and 12 months, postoperatively. Patients with a persistent peripheral graft detachment 4 weeks after DMEK (n=16) were observed for their spontaneous course up to 1 year postoperatively. Results Persistent graft detachments could be characterised into two phenotypes: peripheral roll (n=11; 69%) and laminar detachment (n=5; 31%). Maximal length of the detachment did not change in peripheral rolls during observation period (12 months vs 4 weeks, 578±122 µm vs 593±106 µm, p=0.74), whereas laminar detachments spontaneously attached to the host9s stroma (12 months vs 4 weeks, 0 µm vs 1088±295 µm, p≤0.001). Central corneal thickness and (peripheral) corneal thickness above the detached area did not significantly change in either group. Conclusions Persistent peripheral graft detachments after DMEK occurred in 10% of patients and had two distinct OCT-phenotypes. Peripheral rolls did not change during the first 12 months, postoperatively. By contrast, peripheral laminar detachments attached spontaneously even months after surgery. Corneal thickness reduction was only observed above peripheral laminar detachment, but not above peripheral rolls.

Mucosal surfaces are constantly exposed to pathogens and show high immunological activity. In a broad variety of ocular surface disorders inflammation is common, but underlying mechanisms are often not fully understood. However, the main clinical problem is that inflammatory processes are difficult to characterize and quantify due to the impossibility of repeated tissue probing of the delicate ocular surface. Therefore non-invasive optical methods are thought to have the potential for intravital investigation of ocular surface inflammation. This study demonstrates the general potential of two-photon microscopy to non-invasively detect and discriminate key players of inflammation in the ocular surface by using intrinsic fluorescence-based features without the necessity of tissue probing or the use of dyes. The use of wavelength dependent measurements of fluorescence lifetime, in addition to autofluorescence intensity enables a functional differentiation of isolated immune cells in vitro at excitation wavelengths between 710 to 830 nm. Mixed cell cultures and first in vivo results indicate the use of excitation wavelength of 710 to 750 nm for further experiments and future use in patients. Two photon based autofluorescence features of immune cells enables non-invasive differentiation.

Background/Aims To analyse patients with chronic ocular graft-versus-host disease (GvHD) under treatment with 100% autologous serum eye drops from a sealed manufacturing system. Methods 17 patients with chronic ocular GvHD received 100% autologous serum eye drops from single use vials manufactured in a sealed system. Retrospective analysis included visual acuity, corneal staining, frequency of artificial tears, ocular symptoms by means of a questionnaire and information on subjective side effects and cost compensation. Results Data of prior to autologous serum eye drops therapy and at a 6-month follow-up were obtained. They demonstrated a significant increase in visual acuity (logMAR oculus dexter/right eye (OD) 0.5±0.32 to 0.4±0.3; oculus sinister/left eye (OS) 0.6±0.35 to 0.3±0.35; p=0.177/0.003) and significant improvement in corneal staining (Oxford grading scheme: OD from 3±1.03 to 2±1.43, OS from 4±1.0 to 2±1.09, p=0.004/0.001) and ocular symptoms (ocular surface disease index: 88±20.59 to 63±22.77; p=0.02). Frequency of artificial tears was reduced and no side effects were reported. Patient satisfaction was 100%, and cost compensation by health insurance reached 80%. Conclusions 100% autologous serum eye drops using a sealed manufacturing system were efficient in improving the ocular surface, patient symptoms and visual acuity without side effects. It seems to be safe to use 100% autologous serum despite earlier suspicions regarding immune complex accumulations and exacerbation of ocular surface inflammation. The potential effects of serum levels of systemic immunosuppressives through readministration onto the ocular surface need to be elucidated.

Early and correct diagnosis of delayed or absent corneal epithelial wound healing is a key factor in the prevention of infection and consecutive destruction of the corneal stroma with impending irreversible visual loss. Two-photon microscopy (TPM) is a novel technology that has potential to depict epithelial cells and to evaluate cellular function by measuring autofluorescence properties such as fluorescence intensity and fluorescence lifetimes of metabolic co-factors such as NAD(P)H. Using non-invasive TPM in a tissue-culture scratch model and an organ-culture erosion model, fluorescence intensity and fluorescence lifetimes of NAD(P)H were measured before and during closure of the epithelial wounds. Influence of temperature and selective inhibition of metabolism on intensity and lifetimes were tested additionally. Decrease of temperature resulted in significant increase of fluorescence lifetimes and decrease of the relative amount of free NAD(P)H due to decreased global metabolism. Increase in temperature and upregulation of glycolysis through blocking the mitochondrial electron transport chain by rotenone resulted in increased intensity, decreased lifetimes and increase in the relative amount of free NAD(P)H. Changes of lifetimes and free:protein-bound NAD(P)H ratios were similar to changes measured during wound healing in both scratch and erosion models. Fluorescence lifetime measurements (FLIM) detected enhancement of cellular metabolism following epithelial damage in both models. The prospective detection of cellular autofluorescence in vivo, in particular FLIM of metabolic cofactor NAD(P)H, has the potential to become an indispensible tool in clinical use to differentiate healing from non-healing epithelial cells and to evaluate effects of newly developed substances on cellular metabolism in preclinical and clinical trials.

Human beta-defensins are cationic peptides produced by epithelial cells that have been proposed to be an important component of immune function at mucosal surfaces. In this study, the expression and inducibility of beta-defensins at the ocular surface were investigated in vitro and in vivo. Expression of human beta-defensins (hBD) was determined by RT-PCR and immunohistochemistry in tissues of the ocular surface and lacrimal apparatus. Cultured corneal and conjunctival epithelial cells were stimulated with proinflammatory cytokines and supernatants of different ocular pathogens. Real-time PCR and ELISA experiments were performed to study the effect on the inducibility of hBD2 and 3. Expression and inducibility of mouse beta-defensins-2, -3 and -4 (mBD2-4) were tested in a mouse ocular surface scratch model with and without treatment of supernatants of a clinical Staphylococcus aureus (SA) isolate by means of immunohistochemistry. Here we show that hBD1, -2, -3 and -4 are constitutively expressed in conjunctival epithelial cells and also partly in cornea. Healthy tissues of the ocular surface, lacrimal apparatus and human tears contain measurable amounts of hBD2 and -3, with highest concentrations in cornea and much lower concentrations in all other tissues, especially tears, suggesting intraepithelial storage of beta-defensins. Exposure of cultured human corneal and conjunctival epithelial cells to proinflammatory cytokines and supernatants of various bacteria revealed that IL-1beta is a very strong inductor of hBD2 and Staphylococcus aureus increases both hBD2 and hBD3 production in corneal and conjunctival epithelial cells. A murine corneal scratch model demonstrated that beta-defensins are only induced if microbial products within the tear film come into contact with a defective epithelium. Our finding suggests that the tear film per se contains so much antimicrobial substances that epithelial induction of beta-defensins occurs only as a result of ocular surface damage. These findings widen our knowledge of the distribution, amount and inducibility of beta-defensins at the ocular surface and lacrimal apparatus and show how beta-defensins are regulated specifically.

Purpose To evaluate the use of 2-photon microscopy (TPM), which excites tissue autofluorescence, in detecting and calculating the grade of collagen corneal crosslinks, which are not visible through the slitlamp and in vivo confocal microscopy. Setting Departments of Ophthalmology, University of Lubeck, Lubeck, and University of Rostock, Rostock, Germany. Design Experimental study. Methods Corneas of rabbits were treated with different crosslinking (CXL) protocols. Two weeks after treatment, the corneas were evaluated in vivo by confocal microscopy. Eyes were enucleated and TPM was performed at 710 nm and 826 nm excitation wavelengths to detect tissue autofluorescence, second harmonic generation, and fluorescence lifetime measurements (FLIM). Eyes were then fixed and analyzed by histology. Results Crosslinking following the standard protocol generated a strong autofluorescence signal in the stroma that was detected by TPM. This signal was weakly present in the control specimens, and a sharp transition zone between the peripheral zone and the CXL zone was seen. On FLIM, an increase in corneal crosslinks was measured when the standard protocol was used. Conclusions Two-photon microscopy, a noninvasive method, was able to detect the effects of therapeutic CXL and measure the grade of CXL. In addition to postoperative treatment control, the technique has possibilities for use in online dosimetry during 2-photon triggered CXL. Financial Disclosure No author has a financial or proprietary interest in any material or method mentioned.

The subglottic region of the larynx is of high clinical relevance with regard to infections and malignancies. Little is known about the distribution of mucins and antimicrobial substances in this area. In this study, we have investigated the mucin distribution in the normal subglottis of the larynx. Moreover, we analysed the expression of lysozyme and lactoferrin in this area. Therefore, the subglottic region of 34 larynges was investigated immunohistochemically with different antibodies to mucins and antimicrobial substances. The epithelium reacted positive with antibodies to mucins MUC1 (34/34), 5AC (26/34), 5B (10/34), 7 (8/34), 8 (10/34) and 16 (19/34); submucosal glands were positive to mucins MUC1 (34/34), 5B (10/34), 7 (8/34), and 16 (19/34); high columnar epithelial cells and serous parts of subepithelial seromucous glands were also positive for lysozyme (34/34) and lactoferrin (34/34). The results show that human subglottic epithelium and subepithelial submucosal glands produce a broad spectrum of mucins that is almost comparable with that in other areas of the respiratory tract. We hypothesize that the mucin diversity of the subglottis has an impact on positive functional consequences during vocal production and antimicrobial defence. This antimicrobial defence is supported by synthesis and secretion of antimicrobial substances such as lysozyme and lactoferrin. Moreover, knowledge of the observed distribution pattern of mucins in the subglottis can be a useful tool for a classification of subglottic laryngeal carcinomas.

sumed to be a key location for the generation of adaptive immune mechanisms of the ocular surface, but functional studies of CALT are still lacking. The purpose of this study was to establish an animal model that enables functional analysis of immune mechanisms going on within CALT. In addition, the use of two-photon microscopy, a new optical method, was evaluated for examining complex immunologic interactions of CALT by volume (three-dimensional [3-D]) and time-dependence (four-dimensional [4-D]) in vivo. METHODS. The conjunctiva of female BALB/c mice was repeatedly challenged with topical Chlamydia trachomatis serovar C or a solution of ovalbumin and cholera toxin B. Two-photon microscopy was conducted on explanted, unfixed, and unstained eyes with adjacent nictitating membranes. RESULTS. After three to five stimulations, CALT was detected exclusively in the nictitating membrane of 73% (C. trachomatis) or 70% (ovalbumin/ cholera toxin) of the animals. CALT mainly consisted of CD45R/B220 B cells and CD4 and CD8 T cells. Electron microscopy showed intraepithelial lymphocytes and follicles consisting of lymphocytes, dendritic cells, and macrophages. Two-photon microscopy based on tissue autofluorescence allowed all components of CALT to be detected three dimensionally. High-resolution images were generated in tissue depths of 65 m below the mucosal surface. CONCLUSIONS. This study introduces a novel mouse model for functional investigations of CALT. Topical stimulation with C. trachomatis or ovalbumin/cholera toxin B reliably leads to CALT generation at the nictitating membrane. The use of twophoton microscopy enables groundbreaking 3-D and, in the future, intravital 4-D investigations of immunologic processes initiated in CALT. (Invest Ophthalmol Vis Sci. 2008;49: 1512‐1517) DOI:10.1167/iovs.07-0809

Background: Due to laryngeal neoplasia, as well as infectious and autoimmune diseases, the subglottic region is of great clinical relevance. However, descriptions of the subglottic structures are inconsistent. The aim of our study was to present a precise analysis of the subglottic region and derive functional and clinical conclusions. Methods: Histological, histochemical and immunohistochemical investigations as well as scanning electron microscopy were performed and combined with injection techniques applied to the subglottic region of the larynges of 33 body donors. Results: The three-dimensional extensions of the subglottic region were newly defined: the inferior arcuate line of the vocal cord was defined as the cranial border. The lower margin of the cricoid is the caudal border. Craniolaterally, the elastic cone and, further caudally, the cricoid form the border. Therefore, the definition presented comprises heretofore unnamed ventral and dorsal parts of the lower larynx. The subglottic region can be described as cylindrical, becoming smaller in the cranial direction, following the elastic cone. The ventral boundary is formed by the median part of the cricothyroid ligament (ligamentum conicum), the dorsal part by the cricoid cartilage. The walls of the subglottis are divided into three or four layers composed of collagenous and elastic fibres in which seromucous glands are embedded. Subglottic blood vessels including a tight subepithelial capillary plexus were delineated. Conclusion: The new definition of the subglottic extensions presented is helpful and essential for precise laryngeal tumour classification. The results indicate that the boundaries are unlikely to counteract tumour progression in the subglottis. Furthermore, the findings suggest that these structures contribute to temperature regulation of breath, protection against inflammation as well as breath frequency and depth-dependent mucus secretion.

Aim: To report on a new postoperative finding after delamination of the internal limiting membrane (ILM) for the treatment of different macular pathologies. Methods: Seven patients are described with paracentral retinal holes, all located temporally to the macula, which developed after pars plana vitrectomy and uncomplicated ILM removal for cystoid macular oedema (n = 3), macular pucker (n = 3), and macular hole (n = 1) with the use of indocyanine green (n = 4), trypan blue (n = 1), triamcinolone acetonide (n = 1), or without dye (n = 1). Conclusion: The use of dyes has been incriminated in causing iatrogenic damage to the retina. In this series, the new observation of retinal holes in the macular area is thought to be related to the removal of the ILM itself. The authors speculate that ILM delamination may cause a retinal weakening by Müller cell damage; this may be followed by a structural breakdown and, consequently, hole formation., link_to_OA_fulltext

TFF peptides (formerly P domain peptides, trefoil factors) are typical secretory products of mucin-producing cells and are thought to influence the rheological properties of mucous gels. We investigated the localization of these peptides in the human false vocal folds of the larynx, also known as the ventricular folds or vestibular folds. An analysis of TFF peptide mRNA by RT-PCR and TFF protein by Western blot detected TFF1 and TFF3, but not TFF2. Immunohistochemistry revealed TFF1 to be associated with the secretory product of goblet cells and mucous parts of subepithelial seromucous glands. TFF3 occurred in columnar epithelial cells of the mucosa and in serous cells and excretory duct cells of seromucous glands. These peptides may play a role in the rheological function of mucus secreted onto the true vocal folds and are thus important constituents of vocal production.

Inflammatory processes often lead to pathologic changes in the area of the larynx. A moistening function of the false vocal folds has been described frequently. Up to now we have little knowledge of the role of the false vocal folds in protection against pathogenic agents. The present study analyzes the structures of the false vocal folds in their relations to antimicrobial defense mechanisms. Investigations were performed on false vocal folds of larynges from 34 cadavers using histologic, histochemical and immunohistochemical methods. Seromucous glands, together with epithelial and goblet cells of the folds, synthesize a complex mucus layer. In all of the investigated samples this layer contains carbohydrates including N-acetyl-glucosamine, N-acetyl-galactosamine, galactose, mannose, fucose, and sialic acids. Furthermore, antimicrobial peptides like lactoferrin, lysozyme, alpha and beta defensins are also found in these structures. IgA, produced by plasma cells in the false vocal folds, is frequently integrated in the secretory product. Synthesized mucins, antimicrobial peptides and immunoglobulins form a specialized protective substance that is secreted mainly at the true vocal folds. Here the layer functions to lubricate the true vocal folds, resulting in positive functional consequences during vocal production. Moreover, together with immunocompetent cells, the protective layer seems to play a major role in antigen defense and prevents invasion of pathogenic agents.

Purpose Conjunctiva-associated lymphoid tissue (CALT) is thought to play a key role in initiating ocular surface related immune responses. This study was planned to get first profound insights into the function of CALT related to development, cellular dynamics and morphological alteration using a novel mouse model. Methods Expression and morphology of CALT were investigated using BALB/c mice kept under different housing conditions, after topical antigen-stimulation and following lymphadenectomy and splenectomy. Particles and bacteria were applied topically to study antigen-transport. Intravital visualization was performed using two-photon microscopy. Results Postnatal development and ultrastructure of CALT in the mouse is similar to humans. Topical antigen-challenge significantly alters CALT expression. Bacterial translocation is demonstrated via lymphoepithelium whereas cellular velocities within follicles were approximately 8 µm/min. Conclusions CALT in the mouse is an immunological interface of the ocular surface, featuring dynamic processes such as morphological plasticity, particle/bacteria transport and cellular migration.

A new "orcein-picroindigocarmine staining", a colour combination of orcein, indigo carmine, and picric acid, was developed for histological applications. The new technique was tested on different human tissues. Colours ranging from red to brown, yellow, green and blue were observed in paraffine sections of tissues stained by this method. Nuclear structures in all tissues were stained dark brown to dark blue. Squamous epithelium was stained light brown with varying shades of blue in upper horny layers, whereas the ciliated epithelium was tinged blue grey. When connective tissue was stained, collagen fibrils appeared strongly blue next to elastic fibres, which took on a rust brown tinge; cellular components were all coloured brown. The matrix of hyaline cartilage was stained in different shades of blue, with the chondrocytes rust brown. Sections of bone components appeared dark blue to dark green. Skeletal muscle cells were coloured yellow and green with blue collagenous septa. The new staining is useful for distinguishing connective tissue components such as elastic fibres and collagen fibrils. It also demonstrates chondrocytes in favourable contrast to the cartilage matrix. The technique produces aesthetic staining colouring that could supplement histological investigations and provide an alternative to other staining materials.

Background The role of lymphatic vessels in tissue and organ transplantation as well as in tumor growth and metastasis has drawn great attention in recent years. Methodology/Principal Findings We now developed a novel method using non-invasive two-photon microscopy to simultaneously visualize and track specifically stained lymphatic vessels and autofluorescent adjacent tissues such as collagen fibrils, blood vessels and immune cells in the mouse model of corneal neovascularization in vivo. The mouse cornea serves as an ideal tissue for this technique due to its easy accessibility and its inducible and modifiable state of pathological hem- and lymphvascularization. Neovascularization was induced by suture placement in corneas of Balb/C mice. Two weeks after treatment, lymphatic vessels were stained intravital by intrastromal injection of a fluorescently labeled LYVE-1 antibody and the corneas were evaluated in vivo by two-photon microscopy (TPM). Intravital TPM was performed at 710 nm and 826 nm excitation wavelengths to detect immunofluorescence and tissue autofluorescence using a custom made animal holder. Corneas were then harvested, fixed and analyzed by histology. Time lapse imaging demonstrated the first in vivo evidence of immune cell migration into lymphatic vessels and luminal transport of individual cells. Cells immigrated within 1–5.5 min into the vessel lumen. Mean velocities of intrastromal corneal immune cells were around 9 µm/min and therefore comparable to those of T-cells and macrophages in other mucosal surfaces. Conclusions To our knowledge we here demonstrate for the first time the intravital real-time transmigration of immune cells into lymphatic vessels. Overall this study demonstrates the valuable use of intravital autofluorescence two-photon microscopy in the model of suture-induced corneal vascularizations to study interactions of immune and subsequently tumor cells with lymphatic vessels under close as possible physiological conditions.

Purpose Cyclosporine A (Cs) has been used as effective topical therapy for inflammatory dry eye disease since more than a decade. However, due to its lipophilic character, Cs is formulated as emulsions or oily solutions for topical application. This experimental study aimed to test if the use of semifluorinated alkanes (SFAs) as a preservative-free, well-tolerated non-stinging or burning vehicle maintains or even improves the benefits of Cs in the topical therapy of dry-eye disease. Methods Desiccating stress was applied to C57BL/6 mice for 14 consecutive days to induce experimental dry-eye. Cs dissolved in SFA (perfluorobutylpentane = F4H5with 0.5% Ethanol), F4H5 with 0.5% ethanol only, 0.05% Cs (Restasis®), and dexamethasone (Monodex®) were applied three times daily beginning either at day 4 or day 11 of desiccating stress for up to 3 weeks after end of dry-eye induction. Results In comparison to other groups, Cs/F4H5 demonstrated high efficacy and earlier reduction of corneal staining. In this study, Cs/F4H5 had the ability to maintain conjunctival goblet cell density once applied on day 4. Flow cytometry analysis from cervical lymphnodes demonstrated a significantly lower CD4+ and CD8+ T-cells in the Cs/F4H5 group following 3 weeks of therapy than at baseline, but no difference in regulatory T cells from regional lymphnodes were seen. Conclusions Overall, compared to a commercially available Cs formulation (Restasis®) and dexamethasone, Cs/F4H5 was shown to be equally effective but with a significantly faster therapeutic response in reducing signs of dry-eye disease in an experimental mouse model.